The invention relates to increasing the half-life of circulating therapeutically effective enzymes in blood. As used herein, the term "therapeutically effective enzyme" means an enzyme which, when administered to a human patient, catalyzes a reaction beneficial to the health of the patient.
Therapeutic proteins have been modified in a number of instances to increase the circulatory half-life of the proteins. Davis et al. U.S. Pat. No. 4,179,337 describes covalently coupling proteins with polyethylene glycol to increase the circulation half-life of the protein while retaining enzymatic activity. Rosenblum et al. (1985) Cancer Res. 45, 2421 describes coupling human leukocyte interferon with an anti-interferon monoclonal antibody to increase the interferon's plasma half-life while maintaining its antiviral and antiproliferative properties.
Rijken et al. (1979) Biochim. Biophys. Acta. 580, 140 describes the partial purification, from human uterine tissue, of tissue plaminogen activator (t-PA), a single chain zymogenic enzyme activatable by plasmin in blood to a two-chain form which greatly accelerates the dissolving of fibrin clots. t-PA is thus useful as a therapeutic agent for dissolving clots in patients suffering from a variety of vascular diseases, particularly myocardial infarctions resulting in clots in and around the heart. Wei et al., U.S. Ser. No. 782,686, filed Oct. 1, 1985, assigned to the same assignee as this application and hereby incorporated by reference, describes the use of recombinant DNA techniques to produce human uterine t-PA.
A number of methods have been used to modify t-PA and other fibrinolytic enzymes (e.g., urokinase). Maksimenko et al. (1985) Thromb. Res. 38, 289 describes increasing the affinity of urokinase for fibrin clots by conjugating urokinase to fibrinogen; the resulting complex was subsequently incoporated into a fibrin clot during its natural formation. Maksimenko et al. (1985) Thromb. Res. 38, 277 describes a urokinase-sodium nitroprusside-heparin complex with simultaneous fibrinolytic, hypotensive, and anticoagulant effects. Sevilla et al. (1985) Biochim. Biophys. Res Commun. 130, 91 describes conjugating urokinase to polyclonal anti-human fibrinogen antibody through a bifunctional cross-linking reagent; the resulting conjugate had a strong binding affinity to human fibrinogen while retaining urokinase amidase activity. Ferres et al. E.P.A. 85100084.4 describes coupling t-PA and urokinase to human albumin, immunoglobin G, blocked plasmin, and fibrinogen through bifunctional cross-linking reagents; the resulting conjugates "retain fibrinolytic activity and have slow physiological clearance rates, and/or improved in vivo activity."